1. A Thrombospondin-Dependent Pathway for a Protective ER Stress Response
Jeffrey M. Lynch, Marjorie Maillet, Davy Vanhoutte, Aryn Schloemer, Michelle A. Sargent, N. Scott Blair, Kaari A. Lynch, Tetsuya Okada, Bruce J. Aronow, Hanna Osinska, Ron Prywes, John N. Lorenz, Kazutoshi Mori, Jack Lawler, Jeffrey Robbins, Jeffery D. Molkentin 
Cell 
Volume 149, Issue 6, Pages (June 2012)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Thbs4 Expression Is Upregulated in Diseased Hearts and Is Protective
(A) Western blots for Thbs4 protein expression in different mouse models exhibiting cardiac disease.
(B) Schematic diagram representing the binary transgenic system regulated by doxycycline to inducibly overexpress Thbs4 in the heart.
(C) Representative western blot showing Thbs4 protein expression level in heart extracts from 4-month-old DTG mice versus WT and tTA-only transgenic controls.
(D) Immunohistochemistry for Thbs4 (green) in 4-month-old mouse hearts. Red staining shows membranes, and blue shows nuclei (scale bars = 10 μm).
(E) Hemodynamic assessment of cardiac performance as maximal dP/dt in anesthetized, closed-chested mice in response to increasing doses of the β-adrenergic agonist dobutamine (measured in mmHg/s; ± SD).
(F) Survival plot for tTA and DTG mice following MI injury (n = 10 male mice per group).
(G) Cardiac ventricular fractional shortening (FS%) determined by echocardiography. At least 6 mice were analyzed in each group at 2 and 4 weeks after MI injury in the tTA control and DTG groups (#p < 0.05 versus tTA before MI).
(H) Survival plot in Thbs4−/− and WT mice after pressure overload by TAC (n = 10 male mice per group).
Also see Figures S1 and S2.
Cell  , DOI: ( /j.cell )
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4. Figure 2
Unique ER Stress-Induction Profile Associated with Thbs4 Expression
(A) Western blots for expression of ER stress-response proteins from the hearts of two lines of Thbs4 DTG mice versus tTA control hearts.
(B) Immunohistochemistry for Atf6α and BiP (green) in heart sections from tTA controls or Thbs4 DTG mice. Membranes of the cardiomyocytes are shown in red (scale bars = 10 μm).
(C) Increasing RT-PCR cycles for Atf6α mRNA in hearts of tTA control or Thbs4 DTG mice. Gapdh mRNA was used as a loading/normalization control. The asterisk shows increased Atf6α mRNA in the DTG hearts at the most linear cycle number in the RT-PCR reaction.
(D) Transmission EM of heart sections from tTA control and Thbs4 DTG mice shows ER/SR and vesicles (arrowheads) in control and Thbs4 DTG hearts.
(E) Luciferase activity in the media of NIH 3T3 cells transfected with a plasmid encoding a secreted version of luciferase that was cotransfected with Thbs4, Atf6α-Δ (constitutively nuclear), or Atf4 (all internally normalized to cotransfected β-gal plasmid). ∗p < 0.05 versus the luciferase plasmid only. Error bars represent SD.
(F) Western blotting for the ER processed and secreted proteins Armet and ANF from concentrated media of neonatal rat cardiomyocytes previously infected with adenoviruses encoding β-galactosidase (control), Thbs4, or Thbs1.
Also see Figures S3 and S4.
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5. Figure 3
Thbs4 Induces a Unique ER Stress-Response Signature that Antagonizes Protein Aggregation and Disease in the Heart
(A and B) Western blots showing ER protein expression in (A) αB-crystallin (CryAB) mutant or (B) desmin (DesMut) mutant hearts in comparison to nontransgenic (NTG) and Thbs4 DTG hearts. The hash marks or arrowheads show the different isoforms that were detected.
(C) Immunohistochemistry of frozen heart sections from 6-month-old mice of the indicated genotypes. Protein aggregation (green color) is dramatically reduced when Thbs4 is overexpressed. Red staining is for actin and shows the outline of cardiomyocytes (scale bars = 100 μm).
(D) FS% as determined by echocardiography suggests that cardiac function is improved in both aggregation-prone cardiomyopathic transgenic mouse models when Thbs4 is overexpressed (n = 6 or more mice in each group, ∗p < 0.05 versus tTA; #p < 0.05 versus DesMut; †p < 0.05 versus CryAB).
(E) Western blotting for proteins involved in ERAD from the hearts of Thbs4 DTG mice versus tTA control hearts. The arrowheads show the positions of the relevant proteins, whereas the bracket shows two relevant bands.
(F) Western blotting for proteins involved in autophagy (LC3, both I and II isoforms) or cellular protection.
Also see Figure S5.
Cell  , DOI: ( /j.cell )
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6. Figure 4
Thbs4 Functions from within the Cell to Promote Adaptive ER Stress-Response Signaling
(A) Western blots of ER stress-response protein expression in heart extracts from 3-month-old tTA and DTG mice fed chow containing doxycycline for the indicated periods of time to extinguish Thbs4 expression. “No dox” controls are also shown and were generated from extracts of 4-month-old DTG mice. An N-terminal Atf6α antibody from ProSci was used. The arrowheads show the specific bands that were detected; ns = nonspecific.
(B) Immunocytochemistry for Thbs4 in adenovirus-infected neonatal cardiomyocytes showing that Thbs4 (green) has an ER and Golgi staining pattern. Red staining is for membranes, and blue stains the nuclei (scale bars = 10 μm).
(C) Western blotting for Atf4 and Atf6α protein expression in neonatal rat cardiomyocytes infected for 24 hr with recombinant adenovirus or treated with recombinant Thbs4 (2 μg/ml) or tunicamycin (1 μg/ml).
(D) Western blotting from media for Thbs4 after AdThbs4 infection versus Adβgal control in neonatal cardiomyocytes. Ponceau staining is a control for protein loading between the samples.
(E) Western blotting for ER stress-response proteins from neonatal cardiomyocytes following infection with a control (Adβgal) adenovirus or an adenovirus expressing Thbs4 fused with a KDEL ER retention/relocalization signal.
(F) RT-PCR for Thbs4 or Gapdh in neonatal cardiomyocytes treated for 5 hr with ER stress inducers DTT (5 mM) or tunicamycin (10 μg/ml).
(G) AlamarBlue survival assay in DTT-treated (8 hr, 4 mM) neonatal cardiomyocytes infected with AdThbs4 or Adβgal (assay was run in duplicate in three separate experiments; #p < 0.05 versus vehicle; error bars represent SD).
Cell  , DOI: ( /j.cell )
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7. Figure 5 Thbs Proteins Directly Interact with Atf6α
(A) Western blot following GST-Thbs4 pull-down of the indicated proteins from cardiac protein extracts from Thbs4 DTG hearts. A C-terminal Atf6α antibody from ProSci was used.
(B) Mouse Atf6α schematic diagram showing relative size of the lumenal domains used for GST pull-down experiments (red bars).
(C) Western blot for tagged truncations of two different Atf6α lumenal domains following GST-Thbs4 pull-down. The ER lumenal domain of Atf6β did not interact with GST-Thbs4. There is a nonspecific band in the GST (Atf6α ) only lane due to extreme loading of purified GST.
(D) Western blot for a tagged Atf6α lumenal domain (amino acids 361–656) following GST-Thbs1 pull-down.
(E) Gal4-dependent luciferase reporter activity with the lumenal fragments of Atf6α (amino acid numbers shown) as fusions to full-length Gal4 transiently transfected into HEK293 cells with or without Thbs4. The basal activity of each construct was set to 100, from which Thbs coexpression was compared. ∗p < 0.05 versus no Thbs4 control. Results are averaged from three separate experiments.
(F) Schematic diagram of human Thbs4 with the relative sizes of the GST fragments indicated (red bars). A western blot for the Atf6α lumenal domain with a Flag antibody following GST pull-down with the different Thbs4 domains, depicted below the schematic.
(G) Western blot for Thbs4 and Atf6α (FL, full-length is shown detected with ProSci C-terminal antibody) after Atf6α immunoprecipitation (with N-terminal Abcam antibody) from cardiac extracts of control tTA or Thbs4 DTG hearts.
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8. Figure 6 Thbs4 Is Necessary for Atf6α Activation in the Heart
(A) Immunocytochemistry for Atf6α in neonatal rat fibroblasts with Adβgal (control) or AdThbs4 infection, with or without the S1P inhibitor AEBSF. The arrows show prominent nuclear localization of Atf6α, whereas the arrowheads show Atf6α retention in the Golgi and ER. (scale bars = 10 μm).
(B) Western blots showing ER stress-response protein expression in hearts of WT and Thbs4−/− mice at baseline (sham) or after 48 hr of TAC stress stimulation. Arrowheads show the indicated protein.
(C) Immunohistochemistry for Atf6α following 24 hr TAC or a sham surgical procedure. Nuclear-localized Atf6α (arrowheads) was much more prominent in WT hearts after TAC relative to Thbs4−/− hearts with TAC (scale bars = 10 μm).
(D) Western blots showing ER stress-response protein expression in hearts of WT and Thbs4−/− mice at baseline (sham) or 3 days after MI injury.
(E) Western blot for ER stress-response protein expression from isolated adult myocytes in temporary culture from WT or Thbs4−/− hearts.
(F) AlamarBlue survival assay in tunicamycin-treated adult cardiomyocytes isolated from WT or Thbs4−/− hearts (assay was run in duplicate; ∗p < 0.05 versus vehicle; error bars represent SD).
Also see Figure S6.
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9. Figure 7
Thbs4-Atf6α Interaction Is Necessary for Atf6α Function in ER Stress Induction in Cardiomyocytes
(A) Schematic diagram of the region of mouse Atf6α that was used to make the dominant-negative or decoy adenovirus (red bar).
(B) Western blotting for the indicated proteins from neonatal cardiomyocytes infected with the indicated recombinant adenoviruses. The red-boxed areas show induction of ER stress-response proteins by Thbs4 overexpression, which is attenuated with the Atf6α DN-expressing adenovirus.
(C) Schematic diagram of the region of Thbs4 that was used to make the dominant-negative or decoy adenovirus (red bar).
(D) Western blotting for the indicated proteins from neonatal cardiomyocytes infected with the indicated recombinant adenoviruses. The red-boxed areas show induction of ER stress-response proteins by Thbs4 overexpression, which is attenuated with the Thbs4 DN-expressing adenovirus.
(E) Immunocytochemistry for endogenous Atf6α in cultured neonatal rat fibroblasts 24 hr after infection with the indicated adenoviruses. The arrows show prominent nuclear localization of endogenous Atf6α by AdThbs4 infection, which was blocked with either AdAtf6α(DN) or AdThbs4(DN) (scale bars = 10 μm).
(F) Gal4-dependent luciferase reporter assay with full-length human Atf6α-Gal4 that was cotransfected in HEK293 cells with combinations of plasmids encoding Thbs4 with either Atf6α(DN) or Thbs4(DN). Results were averaged from three independent experiments. ∗p < 0.05 versus reporter alone (control); #p < 0.05 versus Thbs4. Error bars represent SD.
(G) Western blotting for Atf6α (activated form) or Gapdh (control) in neonatal cardiomyocytes infected with the indicated adenoviruses with or without tunicamycin. The Thbs4-DN construct prevents activation of endogenous Atf6α in response to tunicamycin treatment.
(H) AlamarBlue survival assay in WT or Atf6α−/− MEFs with or without AdThbs4 infection. Assay was performed in triplicate. ∗p < 0.05 versus no drug; #p < 0.05 versus WT MEFs with AdThbs4; †p < 0.05 versus WT MEFs with Adβgal with same drug treatment. Error bars represent SD.
Also see Figure S6.
Cell  , DOI: ( /j.cell )
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10. Figure S1
Thbs4 and Thbs1 DTG Mice Show Protein Expression in the ER/SR Compartment of Adult Hearts from Transgenic Mice, Related to Figure 1
(A) Immunohistochemistry of Thbs4 protein from Thbs4 DTG hearts with costaining for caveolin-3 (red) to show the sarcolemma (plasma membrane). The longitudinal sections show Thbs4 protein (green) almost exclusively in the ER/SR compartment without obvious protein aggregates and without localization to the outer sarcolemma. Nuclei are stained blue. Scale bars = 10 μm.
(B) Immunohistochemistry of Thbs4 protein (green) from Thbs4 DTG hearts with costaining for calreticulin (red) to show the ER/SR compartment. Staining is overlapping and shows almost exclusively intracellular localization. Scale bars = 50 μm.
(C) Immunohistochemistry in the adult heart from a sham and TAC-pressure loaded WT mouse, which shows that endogenous Thbs4 (green) localizes inside the cell with minor extracellular areas, but only after induction by TAC stimulation. Scale bars = 50 μm.
(D) Immunohistochemistry of Thbs1 from Thbs1 DTG hearts also shows nearly exclusive localization to the ER/SR compartment in the adult heart. Membranes are red (wheat germ agglutinin-TRITC), and Thbs1 is in green. Nuclei are blue. Scale bars = 10 μm.
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11. Figure S2
Thbs4 DTG Mice Show No Cardiac Abnormalities with Aging or after Pressure Overload Stimulation, Related to Figure 1
(A) H&E staining of cardiac histological sections from 40-week-old tTA and Thbs4 DTG mice show no differences or overt pathology with Thbs4 overexpression. Scale bars = 100 μm.
(B) Heart weight normalized to body weight (HW/BW) at 16 and 40 weeks of age in single transgenic Thbs4 mice, single transgenic tTA mice, or DTG mice that overexpress Thbs4 in the heart (n = 6 or more mice per group, ± SD). No differences were observed.
(C) Factional shortening percentage (FS%) as determined by echocardiography in tTA single transgenic controls or Thbs4 DTG mice before or 12 weeks after pressure overload induced by TAC surgery (n = 6 or more mice per group, ± SD). Sham controls were also used. No loss of function was observed in either group, suggesting that Thbs4 overexpression did not predispose to disease. There were also no significant differences in lethality between the groups subject to TAC.
(D) Survival of Wt and Thbs4−/− mice after MI injury, which showed slightly greater lethality in the Thbs4−/− group. Sham mice showed no lethality (n = 10 or more mice per MI group).
(E) Ventricular fractional shortening (FS) 4 weeks after MI in the indicated groups of mice. There was a trend toward a greater reduction in FS in Thbs4−/− mice that survived MI. ∗p < 0.05 versus Sham.
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12. Figure S3
Cardiac-Specific Thbs4 Overexpression Increases Expression of Atf6α-Dependent ER Stress Genes, but Thbs4 Did Not Affect Other ER Membrane-Bound Transcription Factors that Are Also Processed and Cleaved for Activation, Related to Figure 2
(A) Immunohistochemistry for ER-resident proteins (Calreticulin, Creld2, and Hyou1) in green, showing that expression is greatly enhanced in the Thbs4 DTG hearts versus NTG (or tTA) hearts. Myocyte membranes are shown in red, and nuclei are shown in blue. Scale bars = 10 μm.
(B) Western blots for other ER membrane bound Creb-related bZIP family members (and Srebp bHLH transcription factors) from NTG and Thbs4 DTG hearts shows only activation and upregulation of Atf6α (N terminus, nuclear form), but no other factors examined. There were also no differences in the level of proteases (S1P and S2P) involved in Atf6α processing nor in other processing proteins (Insig1 and Insig2). Thus, the Thbs4 effect on ER stress signaling is highly specific to Atf6α. The Atf6α shown in the western blot corresponds to the cleaved/activated form that enters the nucleus. The western blot for Atf6β was taken from Figure 3A because it was part of the same protein extracts from DTG and WT mice when CryAB mice were also investigated at the same time. Atf6α-N refers to the N-terminal nuclear fragment of Atf6α.
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13. Figure S4
Thbs1 and Thbs4 Induce an Adaptive ER Stress-Response Profile in Heart and Skeletal Muscle of Tissue-Specific Transgenic Mice, Related to Figure 2
(A) Schematic of the binary, inducible transgenic system used to express Thbs1 in the heart.
(B) Western blot for Thbs1 protein from the heart of a Thbs1 DTG mouse not on doxycycline (induced) versus a tTA single transgenic control heart.
(C) Western blots for expression of ER stress response proteins from the heart a single Thbs1 DTG mouse line versus a tTA control heart. FL = full-length Atf6α, N = N-terminal Atf6α fragment.
(D and E) Schematic of the transgene constructs used to make skeletal muscle-specific Thbs4 or Thbs1 transgenic mice and western blots from skeletal muscle of 2 different lines for each Thbs4 and Thbs1 versus a WT control for the indicated ER stress proteins. Gapdh is a loading control. The arrowheads show the specific bands for the indicated proteins.
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14. Figure S5
Thbs4 Reduces Aggregates due to Overexpression of a Mutant CFTR Protein in Cultured MEFs, Related to Figure 3
Immunocytochemistry for CFTR (green) in primary cultured MEFs with costaining for phalloidin (red). MEFs were infected with AdCFTR508 alone or with coinfection of AdThbs4 or Adβgal and 6 days later analyzed. The data show diffuse aggregation (arrows) of the mutant CFTR protein that is reduced with coinfection of AdThbs4. The CFTR antibody used shows nonspecific nuclei staining. Scale bars = 50 μm.
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15. Figure S6
Model for the Proposed Thbs-Dependent Beneficial ER Stress-Response Pathway through Atf6α, Related to Figures 6 and 7
Protein-folding based stress, protein aggregation sensing, or even more general stress to the heart induces Thbs4 expression, which directly controls Atf6α in the ER as it shuttles to the Golgi, where it then facilitates its processing and nuclear translocation. Once Atf6α activity is enhanced it secondarily induces protective chaperones and factors that augment ERAD and autophagy, as well as enhanced secretion, toward resolving stress associated with unfolded proteins or increased demands on protein production with hypertrophic or injury stress to the heart.
Cell  , DOI: ( /j.cell )
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